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Double-Strand Breaks from a Radical Commonly Produced by DNADamaging Agents
Marisa L. Taverna Porro and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
S Supporting Information
*
ABSTRACT: Double-strand breaks are widely accepted to be the most toxic form of
DNA damage. Molecules that produce double-strand breaks via a single chemical
event are typically very cytotoxic and far less common than those that form singlestrand breaks. It was recently reported that a commonly formed C4′-radical produces
double-strand breaks under aerobic conditions. Experiments described herein indicate
that a peroxyl radical initiates strand damage on the complementary strand via C4′hydrogen atom abstraction. Inferential evidence suggests that a C3′-peroxyl radical
induces complementary strand damage more efficiently than does a C4′-peroxyl
radical. Complementary strand hydrogen atom abstraction by the peroxyl radical is
efficiently quenched by thiols. This mechanism could contribute to the higher than expected yield of double-strand breaks
produced by ionizing radiation.
■
INTRODUCTION
Double-strand breaks (dsb) are considered to be one of the
most toxic forms of DNA damage that threaten the integrity of
the genome and cell death.1,2 Failure to repair even a single dsb
can be cytotoxic.3 Although there are many molecules that
cleave nucleic acids, the size of the human genome (∼3 billion
base pairs) makes the probability that two molecules acting
independently on DNA will produce a dsb low. Consequently,
molecules and chemical mechanisms that lead to dsbs are of
great interest. We recently identified a mechanism for dsb
formation that could arise from a single chemical reaction with
DNA and involves radical transfer from one strand to another
(Schemes 1 and 2).4 The mechanism of this unusual process is
expanded upon in this article.
Calicheamicin and C-1027 are examples of rare molecules
that directly produce dsbs by abstracting hydrogen atoms from
opposite strands within duplex DNA.5,6 Most recently, another
natural product, lomaiviticin A, has been shown to produce
dsbs.7 A single molecule of bleomycin can also produce dsbs via
a mechanism in which the iron-containing antibiotic is
reactivated following oxidation of one DNA strand while it
remains bound to the duplex.8−11 Several other structurally
related antitumor antibiotics exist that produce bistranded
lesions that can be converted to dsbs.12−14 The bistranded
lesions produced are examples of clustered lesions (2 or more
lesions within ∼1.5 turns of duplex DNA) that are converted to
dsbs as a result of DNA repair or by interactions with amines,
such as those present in the histone proteins within
nucleosomes.15−21
Although ionizing radiation can produce dsbs via two
hydroxyl radicals reacting with opposite DNA strands, this
pathway would be expected to be dependent on the square of
the dose (second order in OH•). However, dsb yield increases
linearly at low ionizing radiation doses. The mechanism put
© 2015 American Chemical Society
forth to explain this phenomenon that is widely accepted is that
multiple OH• (“spurs”) are produced in the vicinity of DNA
due to the ability of the radiation track to ionize several water
molecules.22,23 A second mechanism that has been considered
involves the formation of a radical on one DNA strand by OH•
addition to a nucleobase or abstraction of a hydrogen atom by
it.24,25 A sequence of reactions then ensue resulting in cleavage
of the original strand and formation of a radical on the
opposing DNA strand that ultimately leads to dsb formation.
Chemical support for the latter mechanism was difficult to
attain using ionizing radiation as a source for initiating DNA
damage, possibly due to the large number of reactive
intermediates produced throughout the biopolymer. However,
we recently discovered a process whereby a C4′-nucleotide
radical (1) yields a dsb (Schemes 1 and 2).4 A C4′-radical (1)
was independently generated from a previously reported
photochemical precursor (2, Scheme 2).26 Double-strand
breaks (and bistranded lesions) are produced in an O2dependent manner and are composed of cleavage at the site of
the originally formed radical on one strand and at one of three
nucleotides on the opposite strand (these are indicated by
asterisks in Scheme 1). The three nucleotides cleaved on the
complementary strand are opposite those immediately 5′ to the
position of the originally formed radical. Reaction with the
opposing strand is made possible by cleavage of the original
strand to form a cation radical (3, Scheme 2).26−29 Cation
radical (3) generation is critical for dsb formation due to
concomitant strand scission, which provides the necessary
conformational freedom. Water addition to 3 produces two
regioisomeric radicals (5 and 6, Scheme 2),30,31 which could
yield as many as four diastereomeric peroxyl radicals (7 and 8)
Received: January 19, 2015
Published: March 9, 2015
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DOI: 10.1021/acs.chemrestox.5b00032
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Chemical Research in Toxicology
Scheme 1
Scheme 2
Coulter LS 6500 scintillation counter. Photolyses were carried out in
a Rayonet RPR-100 photoreactor (Southern New England Ultraviolet)
equipped with 16 lamps with maximum emission at 350 nm. BME and
piperidine solutions were freshly prepared.
General Procedure for the Preparation of Modified
Oligonucleotides Containing 2. The syntheses of oligonucleotides
containing radical precursor 2 were carried out on a 1 μmol scale. The
requisite phosphoramidite was prepared as previously described.35 The
standard method for 2-cyanoethylphosphoramidites provided by the
instrument manufacturer was used except that the coupling of the
modified phosphoramidite was extended to 15 min. Deprotection of
the nucleobases and phosphate moieties as well as cleavage of the
linker was carried out using 28% aq. NH3 at 55 °C overnight.
General Procedure for Oligonucleotide Photolysis. The
strands of interest were labeled at their 5′-termini with γ-32P-ATP
using T4 PNK in T4 PNK buffer (70 mM Tris-HCl, pH 7.6, 10 mM
MgCl2, 5 mM DTT, 45 min, 37 °C). Radiolabeled oligonucleotides
were separated from unincorporated 32P-nucleotides by gel filtration
using Sephadex G-25. Prior to photolysis, labeled strands were
hybridized to the complementary strand(s) (1.5 equiv) in PBS (0.1 M
NaCl, 10 mM sodium phosphate, pH 7.2) by heating at 90 °C for 5
min and slow cooling to room temperature. DNA was photolyzed
(350 nm) for 4−10 h under aerobic conditions in Pyrex glass tubes (5
mm i.d.).
Postphotolysis Oligonucleotide Treatments. Aliquots were
treated with NaOH (0.1 M, 30 min, 37 °C; neutralized with 0.1 M
HCl) or piperidine (1 M, 30 min, 90 °C). Piperidine-treated samples
that may carry out interstrand hydrogen atom abstraction.
Peroxyl radicals 7 and 8, or 4 (which is formed reversibly from
1), yield either 3′-phosphoglycolate (PG) or C4-AP.32,33 Mass
spectral analysis of the cleavage products in the complementary
strand suggested that the respective C4′-hydrogen atoms were
abstracted from the corresponding nucleotides in the opposing
strand.4 Whether this pathway was a major contributor and
how fast the process occurred were unknown. In addition, the
identity of the reactive species responsible for complementary
strand damage was uncertain.
■
EXPERIMENTAL PROCEDURES
General Methods. Oligonucleotides were synthesized via standard
automated DNA synthesis on an Applied Biosystems Inc. model 394
instrument. DNA synthesis reagents were obtained from Glen
Research. Oligonucleotides were purified by 20% denaturing gel
electrophoresis and desalted using C18-Sep-Pak cartridges. Oligonucleotides were characterized by ESI (Thermoquest LCQ Deca) or
MALDI-TOF (Bruker Autoflex III) mass spectrometry.
Radiolabeling was carried out using standard protocols and is briefly
described below.34 T4 polynucleotide kinase (PNK) was purchased
from New England Biolabs. γ-32P-ATP was purchased form
PerkinElmer. C18-Sep-Pak cartridges were obtained from Waters.
Quantification of radiolabeled oligonucleotides was carried out using a
Molecular Dynamics phosphorimager equipped with ImageQuant TL
software. Radiolabeled samples were counted using a Beckman
811
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were frozen (dry ice/acetone) and evaporated to dryness under
vacuum. The residue was taken up in H2O (50 μL), vortexed, spun
briefly, frozen, and evaporated to dryness under vacuum. Samples were
analyzed by dissolving in formamide loading buffer prior to separating
by denaturing PAGE.
labeled) reveals visibly significant reduction in direct strand
scission and alkali-labile lesion formation at T50 in the latter
(Figure 2). The product KIEs were estimated by normalizing
■
RESULTS AND DISCUSSION
Isotopic Labeling Detection of Interstrand C4′-Hydrogen Atom Abstraction. Cleavage products from the
complementary strand from which 1 was initially generated
containing 3′-phosphoglycolate termini (PG, Scheme 2) were
detected by LC/MS.4 This was strong evidence for C4′hydrogen atom abstraction en route to dsbs. However, it did
not provide quantitative information regarding the contribution
of C4′-hydrogen atom abstraction to overall dsb formation.
More insight into the overall contribution of C4′-hydrogen
atom abstraction from the complementary strand was gleaned
from experiments in which deuterium was selectively
incorporated at the C4′-position of the nucleotide, which is
the major cleavage site on the complementary strand. It was
anticipated that deuteration of the C4′-position would give rise
to a kinetic isotope effect (KIE), resulting in reduced cleavage
at the deuterated nucleoside.36−40 The reduction in the amount
of strand scission at the nucleotide would be proportional to
the KIE for the hydrogen atom abstraction reaction and the
fraction of overall cleavage at the nucleotide attributable to this
process and are referred to here as product KIEs.38
The C4′-position of the nucleotide (T50), which is the major
site where strand damage is induced in the complementary
strand of 9a (Figure 1), was deuterated (9b).4 Denaturing
PAGE analysis following photolysis of 5′-32P-c-9a and -9b (c
indicates that the strand complementary to the one containing
2 is 32P-labeled; k indicates that the strand containing 2 is 32P-
Figure 2. Autoradiogram of cleavage in 5′-32P-9a and 5′-32P-9b
following photolysis with no further treatment (direct strand scission),
NaOH treatment, or piperidine treatment.
the cleavage at T50 in 5′-32P-c-9a and -9b using the strand
scission at A51. This method removed the extent photochemical
conversion as a variable from the calculation. The values
calculated are the averages of two experiments, with each
containing three samples. The error in the product KIE for
direct strand scission is greatest because the absolute amount of
cleavage is closest to background in these samples (particularly
at 4′-2H-T50 in 9b) and the percentage variation is greatest.
Although the magnitudes of product KIEs are similar when
direct strand scission (3.9 ± 1.9) or NaOH (3.4 ± 0.8) induced
cleavage is measured, the isotope effect is significantly smaller
following piperidine cleavage (1.7 ± 0.3). Direct and NaOHinduced strand scission detect damage resulting from oxidation
of the carbohydrate backbone.41 The cleavage observed upon
NaOH treatment encompasses direct strand breaks and
oxidized carbohydrates that are labile to the alkali. That the
product KIEs under these conditions are within experimental
error of one another suggests that hydrogen atom abstraction
from other positions that give rise to NaOH-labile lesions (e.g.,
C1′), from which hydrogen atom abstraction would not be
affected by C4′-deuteration, do not contribute significantly to
damage on the complementary strand.42 Furthermore, the
small amount of direct and NaOH-labile cleavage observed at
4′-2H-T50 in 9b suggests that abstraction of this hydrogen is a
major contributor to damage resulting from carbohydrate
oxidation at this nucleotide. In contrast, 4′-2H-T50 has a smaller
effect on strand scission at this nucleotide following piperidine
treatment of photolyzed 5′-32P-c-9b. This suggests that there is
one, or more, pathway(s) that does not involve C4′-hydrogen
atom abstraction, which yields damaged products on the
Figure 1. DNA substrates used in this study.
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(identified below), and kDam is the rate constant for
complementary strand damage by the same reactive intermediate(s).43 The slopes of the respective plots for the
dependence of direct strand scission (Figure 3A) and NaOHlabile strand scission (Figure 3B) on GSH concentration were
within error of one another and were approximately 2-fold
greater than those when piperidine cleavage (Figure 3C) was
measured. Comparable observations were made when BME
was used (Figure S1). Assuming that the direct strand breaks
and alkali-labile lesions are derived from a common
intermediate that reacts with the thiols, the decreased kTrap/
kDam ratio is consistent with the approximate doubling of the
total strand scission upon piperidine treatment compared to
that with NaOH reported previously.4 This also suggests that
the rate constant for hydrogen atom abstraction from the 2′deoxyribose ring is approximately equal to that for nucleobase
addition.
complementary strand that are labile to piperidine treatment.
Previously, this property was attributed to nucleobase damage,
which would not be affected by C4′-deuteration, and the
reduced product KIE is consistent with this proposal.4,41
Effects of Thiols on Double-Strand Break Formation.
Product and isotope labeling experiments indicate that peroxyl
radical mediated interstrand C4′-hydrogen atom transfer from
the complementary strand is the major pathway for dsb
formation. However, neither the peroxyl radical (e.g., 7, 8) nor
the efficiency/rate of this process are known. Insight into the
latter was obtained by examining the effects of thiols
(glutathione, GSH; β-mercaptoethanol, BME) on complementary strand damage. The respective form of damage (direct
strand break, NaOH labile cleavage, piperidine labile cleavage)
was measured as a function of thiol concentration (0−300 μM).
The amount of trapping by thiol was estimated to be the
difference in the amount of uncleaved DNA (5′-32P-c-10) in
the presence and absence of the reducing agent. Plotting the
ratio of the trapped intermediate versus cleavage as a function
of thiol concentration (eq 1) yielded a straight line (GSH
(Figure 3); BME (Figure S1)). The slope of these plots
represent the ratio of kTrap/kDam, where kTrap is the rate constant
for reaction between the thiol and reactive intermediate(s)
k Trap
[Trapped]
[GSH]
=
[Cleaved]
kDam
(1)
The response of strand damage to thiol concentration also
provides insight with respect to the species that reacts with the
reductant. The thiols can, in principle, quench complementary
strand damage by intercepting the originally formed C4′-radical
(1) or peroxyl radical(s) (7 and 8). Trapping of 1 in 5′-32P-k10 is not considered to be viable because neither thiol is
expected to compete (k = 2.2 × 106 M−1 s−1) at these
concentrations with O2 (0.2 mM, k = 2 × 109 M−1 s−1), which
reacts reversibly with the nucleotide radical.29 Peroxyl radical
reduction by the thiols is expected to more effectively compete
with hydrogen atom abstraction by these reactive intermediates.29,44 If one assumes that hydrogen atom abstraction from
thiol by a DNA peroxyl radical(s) (7, 8) occurs with rate
constant kTrap = 2 × 102 M−1 s−1,45 then the rate constants for
complementary strand cleavage (kDam) range from ∼1−2 ×
10−2 s−1 (Table 1). On the basis of the previously described
Table 1. Estimated Rate Constants for Complementary
Strand Damage in 5′-32P-10
kDam (×10−2 s−1)a
treatment
GSH
BME
noneb
NaOHc
piperidined
0.8 ± 0.4
1.1 ± 0.5
1.8 ± 0.8
0.9 ± 0.1
1.3 ± 0.1
1.9 ± 0.1
Rate constants are the average ± SD of two independent experiments
each carried out in triplicate. bDirect strand scission. c0.1 M NaOH, 37
°C, 30 min. d1.0 M piperidine, 90 °C, 30 min.
a
products in the complementary strand and what is known
about direct strand scission from various 2′-deoxyribose
radicals, we propose that C4′-hydrogen atom abstraction by 7
and/or 8 is the rate-determining step.4,46 Although there are
not many examples of inter- or intrastrand hydrogen atom
abstraction events by nucleic acid peroxyl radicals that have
been characterized kinetically, the above estimated rate
constant is 2−5-fold faster than C1′-hydrogen atom abstraction
by 17.47
Figure 3. Effect of GSH on cleavage in 5′-32P-10 following photolysis
with (A) no further treatment (direct strand scission), (B) NaOH
treatment, or (C) piperidine treatment.
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Scheme 3
Identifying the Peroxyl Radical(s) Responsible for
Interstrand Damage. Complementary strand damage
requires cleavage within the strand in which the C4′-radical
(1) is originally generated. This provides the conformational
flexibility to react with nucleotides in the complementary
strand. Formation of the nicked strand from 1 proceeds
through the cation radical (3) and ultimately provides two
regioisomeric peroxyl radicals (7 and 8). Preferential formation
of 4′-peroxyl radical (7) is expected.30,31 Insight into the
relative contributions of 3′-peroxyl (8) and/or 4′-peroxyl (7)
radicals to interstrand damage was gleaned by comparing the
reactivity of DNA substrates in which the C4′-radical precursor
(2) is either within an intact strand (e.g. 11) or at the 3′terminus of an otherwise identical ternary complex (e.g. 12).
Substrates containing 2 at the 3′-terminus of an oligonucleotide
within a ternary complex do not produce cation radical 3 and
can form only diastereomeric 4′-peroxyl radicals (7) by reacting
with O2 (Scheme 2). We previously reported that complementary strand damage is enhanced in substrates containing a
stable abasic site analogue (F), presumably by reducing the
barrier for the required conformational reorganization.4 For
instance, complementary strand damage in 11 was ∼2−3-fold
greater than that in 10 (Table 2). However, in a side-by-side
■
CONCLUSIONS
Hydrogen atom abstraction from the C4′-position of
nucleotides is common among DNA damaging agents that
bind in the minor groove.46 Diffusible species, such as hydroxyl
radical, also react at this site because of the relatively low
carbon−hydrogen bond dissociation energy and high solvent
accessibility.38 Double-strand break formation from an initially
formed C4′-radical under aerobic conditions verifies general
mechanistic proposals based upon ionizing radiation studies
and unveils a possible pathway for designing molecules that
produce this type of DNA damage via a single chemical
event.24,25 The experiments described above provide mechanistic insight into how a dsb is produced from abstraction of a
single hydrogen atom from one strand of DNA. Isotopic
labeling reveals that the C4′-hydrogen atom is the major site of
reaction on the complementary strand. Experiments with
ternary complexes containing a C4′-radical precursor at the
3′-terminus of an oligonucleotide further substantiate the
importance of conformational freedom for interstrand hydrogen atom abstraction and suggest that a 3′-peroxyl radical (8)
more efficiently reacts with the complementary strand than
does a regioisomeric C4′-peroxyl radical (7). Competitive
kinetic experiments using thiols reveal that interstrand
hydrogen atom transfer by a peroxyl radical(s) is slow and
would be quenched by physiologically relevant levels of
reductant (millimolar). The rate constants estimated for
interstrand hydrogen atom transfer by the peroxyl radical are
consistent with their expected reactivity. These experiments
suggest that dsb formation emanating from a single initial
hydrogen atom abstraction on one DNA strand is possible but
would be more practical if one could utilize a radical trap other
than O2, which produces a more reactive intermediate that
carries out interstrand hydrogen atom transfer more efficiently.
Table 2. Effect of the Ability To Form Cation Radical 3 on
Complementary Strand Damage
% complementary strand cleavagea
treatment
substrate
form 3?
10
11
12
13
14
15
16
yes
yes
no
yes
no
yes
no
none
3.7
7.6
2.3
0.4
±
±
±
±
b
0.5
2.3
0.3
0.1
1.6 ± 0.5
NaOHc
5.5
14.8
5.3
0.8
0.5
2.5
0.6
±
±
±
±
±
±
±
0.8
1.8
0.4
0.2
0.1
0.8
0.1
piperidined
9.9
20.1
8.5
6.6
4.5
13.0
10.6
±
±
±
±
±
±
±
1.5
2.4
0.9
0.6
1.0
2.5
0.4
Cleavage is the average ± SD of three independent measurements.
Direct strand scission. c0.1 M NaOH, 37 °C, 30 min. d1.0 M, 90 °C,
30 min.
a
b
■
comparison, the complementary strand damage in 12 was
∼2.5−3.5-fold lower than that in 11. The same pattern was
evident, albeit to a lesser extent, when analogous substrates
containing 5′-dGGG sequences (13−16) were photolyzed.
These sequences produce lower levels of sugar damage than
those containing AT sequences. However, the amounts of all
types of complementary strand damage are reduced in ternary
complexes, which cannot form radical cation 3 (14 and 16)
compared to duplexes that can (13 and 15) (Table 2). These
data suggest that the anticipated minor regioisomeric peroxyl
radicals (8) are more effective at inducing complementary
strand damage than are C4′-peroxyl radicals (7). One possible
reason for this is that hydration at C4′-of the cation radical
yields a hemiacetal (8), which can equilibrate under the
aqueous conditions with an acyclic peroxyl radical (18) that
enjoys greater conformational freedom (Scheme 3).
ASSOCIATED CONTENT
S Supporting Information
*
Mass spectra of oligonucleotides containing nonnative
nucleotides. Plots of the effect of BME on complementary
strand damage. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 410-516-8095. Fax: 410-516-7044. E-mail:
[email protected].
Funding
We are grateful for support of this research from the National
Institute of General Medical Science (GM-054996).
Notes
The authors declare no competing financial interest.
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■
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ACKNOWLEDGMENTS
We thank Dr. Arnab Rudra for his assistance with obtaining
ESI-MS data.
ABBREVIATIONS
double strand break, dsb; hydroxyl radical, OH•
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